So-called biotechnology-based pharmaceutical products have been approved, including erythropoietin, or monoclonal antibodies as represented by infliximab, bevacizumab, trastuzumab and adalimumab. These are glycoproteins wherein N-glycans linked to a protein. Immunoglobulin classes of antibodies used in cancer therapy are typically IgG. When the IgG binds to an antigen expressed on a cancer cell, various cascades are turned on to exert anticancer activity which includes 1) antibody-dependent cellular cytotoxicity (ADCC); 2) complement-dependent cytotoxicity (CDC); and 3) a change of signal transduction.
An asparagine at position 297 (N297) in the CH2 domain of the Fc region of an intact Ig antibody is attached to heterogeneous N-glycans. Said N-glycans are mainly biantennary complex type N-glycans having various N-glycan structures. It has been known that this N-glycan is essential for activation of the effector function by an immune complex, and that the activity changes depending on a structure of the N-glycan. For instance, it has been known that lack of α1-6 linked fucose (hereinafter also referred to as a “core fucose”) increases ADCC activity mediated by FcγRIIIa (Shinkawa, Toyohide, et al., J. Biol. Chem., January 2003; 278: 3466-3473). It has been reported that the ADCC activity of fucose-depleted trastuzumab is at least 50 times higher than that of core fucose-linked trastuzumab, and the same results have been reported regarding rituximab, anti-CCR4 antibody, etc. (Niwa, Rinpei, et al., Cancer Res. 64: 2127-2133 (2004)).
In general, Escherichia coli, which are commonly used as a means for producing biotechnology-based pharmaceutical products, do not attach a N-glycan to a protein. Thus, yeast and the like have been used in the production of antibodies. However, a yeast is considered to be problematic because a yeast does not attach the same N-glycan as human but attaches a so-called high mannose type N-glycan. A method of production using insect cells has been proposed, but the same problems with a yeast is concerned. From these reasons, at present, most of antibody drugs are produced by mammalian cells (CHO cells, NSO cells, etc.) that are able to attach a human-type N-glycan, but αGal moiety in the N-glycan attached by NS0 cells has antigenicity. It has been known that N-glycans of an antibody produced by CHO cells or the like is biosynthesized by glycosyltransferase, and that a structure and an amount of the made N-glycan changes depending on number of passage even though the same cell line used. Hence, glycoproteins produced by CHO cells or the like have a problem that the glycoproteins are heterogeneous in N-glycan level, although they are homogeneous in an amino acid sequence level. Moreover, it has been reported that types and abundance ratios of a N-glycan structure linked to a Fc of IgG in human serum is completely different from that of a N-glycan structure linked to Fc produced by CHO (Yamane-Ohnuki, N., et al., Biotechnol Bioeng 87: 614-622 (2004)).
In the case of the N-linked glycans, various types of N-glycans exist as combination of various characters such as a high mannose type N-glycan, a biantennary complex type N-glycan and a complex type N-glycan; the presence or absence of sialic acid (Sia); a difference in the linkage manner; the presence or absence of core fucose; the presence or absence of branched N-acetylglucosamine (GlcNAc); and the like. Accordingly, all of commercially available antibody drugs include a large number of N-glycan structures, and their quality maintenance has been an important issue. The heterogeneity of N-glycan structures make it difficult to approve biosimilars as identical ingredients, and erythropolis has been permitted as a different ingredient name such as Epoetin Kappa, for example.
In the study of N-glycan linked to an antibody and its effect on activity or stability of the antibody by using an antibody having truncated N-glycans revealed that the N-glycan has an effect on the structural stability and activity of the CH2 domain (Mimura, Y., et al., MoI Immunol 37: 697-706 (2000), and Krapp, S., et al., J MoI Biol 325: 979-989 (2003)). Furthermore, a N-linked glycan with branched GlcNAc and the removal of core fucose has been known as a example of preferable characteristic of a N-glycan structure for a pharmaceutical. However, most of commercially available antibody drugs include such N-glycans in only several percentages.
These results of analysis on the function of a N-glycan structure in an antibody give rise to need to produce an antibody having a homogeneous N-glycan structure that imparts preferable characteristics as pharmaceuticals to the antibody. Moreover, it has been also desired to produce an antibody having a homogeneous N-glycan structure in order to achieve pharmaceutical grade quality control.
It has been reported that a genetic polymorphism of FcγR influences on ADCC activity, and that among patients administered with rituximab, those with V/V homozygote at the amino acid position 176 of FcγRIIIa has a higher affinity to IgG1 and IgG3 than those with F/F homozygote (Wu, J., et al., J Clin Invest, 100 (5): pp. 1059-70 (1997)). Furthermore, a patient having a homologous chromosome 158V/158V of FcγRIIIa and a patient having a homologous chromosome 131H/131H of FcγRIIa show higher reactivity with rituximab than a patient having a 158F gene of FcγRIIIa or a patient having a 131R gene of FcγRIIa (Weng, W. K. and R. Levy, J Clin Oncol, 21 (21): pp. 3940-7 (2003)). Further, it has been reported that the 158 V/V genotype of FcγRIIIa has a higher progression-free survival rate than V/F and F/F genotypes in vaccine therapy using an anti-idiotype antibody, (Weng, W. K., et al., J Clin Oncol, 22 (23): pp. 4717-24 (2004)). As such, it has been known that the effects of antibody drugs depend on the genetic polymorphism of FcγR. However, there have been no reports regarding a N-glycan structure giving optimal effects depending on the genetic polymorphism of FcγR.
Thus, it has been desired to produce an antibody having a homogeneous N-glycan structure in an analysis of a preferred N-glycan structure.
Various studies have been conducted to control N-glycans linked to an antibody. For example, a truncated antibody has been examined for determining its effect on attachment of a N-glycan to N297 or on structure of N-glycan (Lund, J., et al., Eur J Biochem 267: 7246-7257 (2000)). In addition, it has been reported that a N-glycosylation pattern is different depending on the type of a cell used for production of an antibody (Lifely, M. R., et al., Glycobiology 5: 813-822 (1995)).
Moreover, studies on β (1,4)-N-acetylglucosaminyltransferase III (GnTIII) that catalyzes the formation of branched GlcNAc have been revealed the optimal expression level of GnTIII for the ADCC activity of an antibody (Umana, P., et al., Nat Biotechnol 17: 176-180 (1999)), and that a co-expression of GnTIII in cells producing a large amount of antibody induces production of an antibody having branched GlcNAc, and the antibodies produced thereby kills target cells at 10 to 20 times lower concentrations than the antibodies produced under no expression of GnTIII (Davies, J., et al., Biotechnol Bioeng 74: 288-294 (2001)).
Knockout of α-fucosyltransferase such as FUT8 for removing core fucose has been disclosed (Yamane-Ohnuki, N., et al., Biotechnol Bioeng 87: 614-622 (2004)). However, this method controls only the core fucose and does not provide an antibody having a homogeneous N-glycan structure. The endoglycosidase S (EndoS, Endo-S, or endo-S) (J. J. Goodfellow, B. G. Davis et al. J. Am. Chem. Soc., 134, 8030-8033 (2012)) and a mutant form thereof (W. Huang, Lai-Xi Wang et al, J. Am. Chem. Soc., 134, 12308-12318 (2012), and WO 2013/120066) have been reported as endoglycosidases that hydrolyze N-glycans in an antibody. However, it has been known that these endoglycosidases recognize a N-glycan structure and have specificity to a specific structure of IgG. Hence, EndoS recognize and hydrolyze specific structures of N-glycans linked to antibodies produced by CHO cells, which leads to a problem of remaining antibodies having unreacted N-glycans as impurities. J. J. Goodfellow, B. G. Davis et al. J. Am. Chem. Soc., 134, 8030-8033 (2012) discloses, a method for preparing an acceptor antibody GlcNAc-Asn-Rituxan which is aimed for removing core fucose, wherein Rituxan produced by CHO cells is hydrolyzed by EndoS, which is then reacted with α-fucosidase from bovine for 20 days. However, this method is so complicated that it is not suitable for industrialization.
On the other hand, in the field of glycoengineering on proteins other than antibodies, there have been various reports. Yamane-Ohnuki, N., et al., Biotechnol Bioeng 87: 614-622 (2004) describes that using endo-β-N-acetylglucosaminidase M (endoglycosidase M, Endo-M, or endo-M) in vitro, N-glycan without fucose was attached to insulin not having N-glycan, and a N-glycoprotein, the monocyte chemotactic protein 3 (MCP-3) was synthesized. However, it has been realized that when an antibody is used as a glycoprotein, a N-glycan locates inside the Fc domain and that make it difficult to completely hydrolyze the N-glycan by Endo-M in contrast to other glycoproteins. Alternatively, other report on glycoengineering on proteins except for antibodies includes a method for producing a glycoprotein having a N-glycan of interest by utilizing a transglycosylation reaction of endoglycosidase, wherein N-glycans in the glycoprotein is hydrolyzed with leaving the GlcNAc at a reducing end so as to prepare an acceptor protein, and then a glycosyl donor of interest is attached to the remaining GlcNAc. This method is described to be used as a method for synthesizing a glycoprotein having any desired homogeneous N-glycan structure by using a glycan derivative having a homogeneous N-glycan structure as a donor. (WO 2007/133855). In order to synthesize a glycoprotein having a homogeneous N-glycan structure by applying a glycan remodeling method, it has been considered that at least three techniques, namely, endoglycosidase (mutant Endo-M, etc.), an oxazoline derivative, and a donor substrate are necessary. It has been reported that, by using these techniques, high mannose type N-glycan conjugated to a RNase B protein can be successfully replaced with a biantennary complex N-glycan to synthesize a glycoprotein having a homogeneous N-glycan structure (W. Huang, Cishan Li et al, J. Am. Chem. Soc., 131, 2214-2223 (2009)).
However, in the case of an antibody remodeling as an acceptor protein, there are several problems such as the conformational location of N297 to be inside the Fc domain inhibits the endoglycosidase to approach to the N-glycan, and when α1-6 fucose is coupled to GlcNAc, the N-glycan cannot be hydrolyzed by Endo-M or the like. Due to these problems, the aforementioned glycoengineering method for proteins other than antibodies cannot be directly applied to antibody glycoengineering. Although there are several reports regarding glycoengineering for antibodies (Roy Jefferis, Nature Review 8: 226-234 (2009)), there have been no reports on a production method that is applicable to commercial production of an antibody having a homogeneous N-glycan structure, i.e. having only N-glycans of interest.